Project Summary/Abstract The human placenta plays a major role in maintaining the proper environment for fetal growth, but remains as the most poorly understood organ. This project aims to substantially improve our knowledge of this important human organ by studying its early development in a systematic, detailed manner, then to combine this knowledge with the latest technologies in regenerative medicine in order to develop in vitro models for the study of both normal and abnormal placental development. Specifically, we aim to understand the mechanisms underlying the establishment and maintenance of a multipotent human trophoblast stem (TS) cell, one which can give rise to all other subtypes of trophoblast, the epithelial cells of the placenta. Over the past few years, we have identified a key pathway, directed by the p53-related protein, p63, which is required for maintenance of undifferentiated cytotrophoblast (CTB) stem cells in the early placenta. More recently, we have noted that a subset of these CTB co-express CDX2, a transcription factor required for maintenance of TS cells in mice, and hypothesize that these CDX2+/p63+ CTB are multipotent human TS cells. We will characterize this subpopulation further, using a combination of FACS sorting, followed by differentiation assays and both bulk and single cell transcriptome profiling. In this discovery-based approach, we will focus our analysis on identification of transcription factors and cell surface markers, which characterize this cell population in the early human placenta. At the same time, we will take a more gene-focused approach, probing the specific role(s) of p63 and CDX2 in first trimester CTB proliferation and differentiation, including their downstream targets. Finally, we will apply this knowledge to human pluripotent stem cells (hPSCs)--both embryonic (hESCs) and induced pluripotent stem cells (hiPSCs)?in order to develop in vitro models for the study of human trophoblast lineage specification and differentiation. We have established a novel differentiation protocol for step-wise differentiation of hPSCs, first into CTB, and subsequently into hCG- secreting syncytiotrophoblast (STB) and HLA-G+ extravillous trophoblast (EVT). Using this protocol, we have found that Trisomy 21 iPSC spend a prolonged period in the CTB stem cell state, and show blunted differentiation into functional STB, identical to the phenotype of primary CTB isolated from placentas with Trisomy 21. These exciting preliminary data suggest that hPSCs may be useful for modeling trophoblast differentiation defects, which are the basis for placental dysfunction. We will compare hPSC-derived trophoblast to primary trophoblast from both pre- and post-implantation tissues in order to determine which they most resemble. The successful completion of this project has the potential to transform the field of human placental biology, by both identifying human TS cells within the placenta, and establishing hPSC-based models of placental disease, thereby constructing a firm foundation on which diagnostic marker discovery and therapeutic targeting of this important human organ would be possible.